Labelling of fusion proteins with synthetic probes

ABSTRACT

The invention relates to new proteins called alkylcytosine transferases (ACTs) derived from O 6 -alkylguanine-DNA alkyltransferase, and to substrates for ACTs specifically transferring a label to these ACTs and to fusion proteins comprising these. The substrates according of the invention are substituted cytosines of formula (I) 
                         
wherein R 1  is an aromatic or a heteroaromatic group, or an optionally substituted unsaturated alkyl, cycloalkyl or heterocyclyl group with the double bond connected to OCH 2 —; R 2  is a linker; and L is a label or a plurality of same or different labels. The invention further relates to methods of transferring label L from these substrates of formula (I) to ACTs and ACT fusion proteins. The system of ACT-compound of formula (I) is particularly suitable for double labelling studies together with the known system O 6 -alkylguanine-DNA alkyltransferase (AGT)-benzylguanines.

CROSS REFERENCE

This application is a divisional of U.S. application Ser. No. 12/309,554filed on Jan. 22, 2009, now U.S. Pat. No. 8,227,602, which is a §371application of PCT Application No. PCT/EP2007/057597 filed Jul. 24,2007, which claims priority from European Patent Application No.EP06117779.6 filed Jul. 25, 2006, herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods of transferring a label fromsubstrates to fusion proteins with a protein part specifically designedto accept the label, to novel specific substrates, and to novel proteinsaccepting the label of specific substrates suitable in such methods.

BACKGROUND OF THE INVENTION

There is a constant need for improved labelling techniques which wouldallow one to specifically label a protein of interest in order toisolate and/or track such protein of interest under in vitro or in vivoconditions. One particular method is disclosed in WO 02/083937describing a method for detecting and/or manipulating a protein ofinterest wherein the protein is fused to O⁶-alkylguanine-DNAalkyltransferase (AGT) and the AGT fusion protein contacted with aspecific AGT substrate carrying a label, whereby the label istransferred to the fusion protein. The AGT fusion protein is thendetected and optionally further manipulated using the label. Severalmutants of wild type AGT were shown to be better suitable than wild typeAGT (WO 2004/031404; Juillerat, A. et al., Chem. Biol. 10:313-317, 2003;Gronemeyer, T. et al., Protein Eng. Des. Sel. 19:309-316, 2006) in sucha labelling method, and a wide range of substituted benzylguanines andrelated heteroarylmethylguanine compounds were described for use intransferring a label to the fusion proteins comprising AGT and AGTmutants (WO 2004/031405).

Simple O²-benzyl-cytosines are known. Freccero, M. et al., J. Am. Chem.Soc. 125:3544-3553, 2003, obtained O²-o-hydroxybenzyl cytosine onreaction of cytosine with o-quinone methide. Ward, A. D. and Baker, B.R., J. Med. Chem. 20:88-92, 1977, describe O²-benzyl cytosine obtainedfrom 2-chloro-4-aminopyridmidine and the sodium salt of benzyl alcohol.

SUMMARY OF THE INVENTION

The invention relates to new proteins called alkylcytosine transferases(ACTs) derived from O⁶-alkylguanine-DNA alkyltransferase, and tosubstrates for ACTs specifically transferring a label to these ACTs andto fusion proteins comprising such ACT. The substrates according of theinvention are substituted cytosines of formula (I)

wherein

R₁ is an aromatic or a heteroaromatic group, or an optionallysubstituted unsaturated alkyl, cycloalkyl or heterocyclyl group with thedouble bond connected to OCH₂—;

R₂ is a linker; and

L is a label or a plurality of same or different labels.

The invention further relates to methods of transferring a label fromthese substrates of formula (I) to alkylcytosine transferases (ACTs) andACT fusion proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: In vitro dual labelling experiments with fusion proteinscomprising AGT and ACT using specific substrates benzylguanine (BG) andbenzylcytosine (BC) carrying fluorescent labels.

Assays were performed by incubating an equimolar mixture of GST-ACT1 and6×His-^(N)AGT with either BGFL, BGCy5, BCFL, BCCy5 or an equimolarmixture of BGCy5/BCFL or BGFL/BCCy5. Fluorescent dye-labelled proteinmixtures were analyzed by SDS-PAGE, see Example 12. GST-ACT1: fusionprotein of ACT 1 (SEQ ID NO:1) with glutathione S-transferase (GST).6×His-AGT: fusion protein of ^(N)AGT (SEQ ID NO:2) with the shortpeptide 6×His. BGFL and BGCy5: benzylguanines substituted with a linkercarrying fluorescein and Cy5, respectively, see Juillerat, A. et al.,Chem. Biol. 10:313-317, 2003, and compound 11 (Example 9). BCFL:compound 5 (Example 4). BCCy5: compound 10 (Example 8).

FIG. 2: Labelling experiments with ACT10 using substrates benzylguanine(BG) and benzylcytosine (BC) carrying fluorescent labels (FL)demonstrating selectivity for BC.

The fluorescent readout of an SDS gel run after different time ofincubation is shown on top with the corresponding graph at the bottom.The percentage of ACT-Fluorescein conjugate (% ACT-FL) formed from ACTis shown vs. time of incubation (min). Assays were performed byincubating 0.5 μM mixture of GST-ACT10 with either BGFL or BCFL.GST-ACT10: fusion protein of ACT 10 (SEQ ID NO:12) with glutathioneS-transferase (GST). BGFL and BCFL: see legend to FIG. 1.

Binding constants found: k_(BC)=1130±150 M⁻¹s⁻¹; k_(BG)˜10 M⁻¹s⁻¹.

FIG. 3: Urea-induced unfolding of ACT1, ACT9 and ACT10.

Shown is the percentage of ACT-Fluorescein (% ACT-FL) formed. The valueobtained at 0 M urea [(NH₂)₂CO] is set to 100%. Protein (0.5 μM) wasincubated in kinetic buffer (50 mM HEPES, pH 7.2, 1 mM DTT) supplementedwith urea (0 to 8 M) for 30 minutes. Then the solution was adjusted to20 μM BCFL and incubated for 2 hours. Samples were then boiled 5 min at95° C. in SDS buffer. Fluorescent dye-labelled protein mixtures wereanalyzed by SDS-PAGE. The data set was fitted with Y=100/(1+10^((logC1/2−X)*HillSlope)) to get the half unfolding urea concentration C1/2.The following values of C1/2 were found for the different mutants:

-   ACT1 2.8±0.1 M; ACT9 4.1±0.1 M; ACT10 5.1±0.2 M.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to new proteins called alkylcytosine transferases(ACTs) derived from O⁶-alkylguanine-DNA alkyltransferase (AGT),particularly suited for the transfer of a label from substrates offormula (I).

An alkylcytosine transferase (ACT) is defined as a protein

(a) consisting of 170 to 220 amino acids, preferably 175 to 190 aminoacids, most preferably 177 to 185 amino acids;

(b) comprising at least one cysteine;

(c) reacting with an O²-benzylcytosine, thereby transferring the benzylsubstituent to the mercapto function of the cysteine of (b) at least asfast as on reaction with an O⁶-benzylguanine under identical conditions.

ACTs of the invention were prepared from DNA encoding AGT in a directedevolution approach based on phage display of AGT. As a starting pointfor directed evolution based on phage display, DNA encoding the mutant^(N)AGT (SEQ ID NO:2, Gronemeyer, T. et al., Protein Eng. Des. Sel.19:309-316, 2006) was used. ^(N)AGT exhibits approximately 50-foldhigher activity towards benzylguanine derivatives than wild-type^(wt)AGT (Juillerat, A. et al., Chem. Biol. 10:313-317, 2003) and haditself been obtained by directed evolution using phage display and abenzylguanine substrate. Codons for residues Tyr114, Lys131, Ser135,Val148, Gly156, Gly157, Glu159 were randomized via saturationmutagenesis. Transformation in phagemid pAK100 resulted in a mutant AGTlibrary of 10⁷ independent clones. For the selection, the phage librarywas incubated with BCFL, a benzylcytosine carrying fluorescein (compound5) as a substrate to label the mutants reacting with benzylcytosine.This allowed for the subsequent enrichment of the corresponding phagesby using magnetic beads covered with anti-fluorescein antibodies. After6 rounds of selection for activity against BCFL, clones ACT1 to cloneACT8 were analyzed by DNA sequencing (Table 1, amino acids shown inone-letter code). In subsequent tests it became obvious that proteinsfrom clone ACT1 to clone ACT8 showed only limited stability againstdenaturation by 4 M urea. To improve this the same library wasre-screened. The protein from the best isolated clone is listed as ACT9and showed good stability in 4 M urea, but limited reactivity towardsbenzylcytosine. Subsequently ACT10 was obtained by error prone PCR ofDNA encoding ACT9 followed by subsequent phage selection resulting infurther modification of residues Met60Ile, Ala121Val and Leu153Ser asthe ACT variant with high reactivity and high stability againstdenaturation by urea. The clones are expressed and the proteins purifiedas glutathione S-transferase (GST) fusion proteins.

These ten proteins specific for benzylcytosine are called alkylcytosinetransferase (ACT) 1 to 10, and are the subject of the present invention.

Further proteins considered in this invention are

-   -   ACTs which are homologs of ACT 1 to 8 and differ from ACT 1 to 8        in one, two or three amino acids in positions other than        positions 114, 131, 135, 148, 157, and 159; and    -   ACTs which are homologs of ACT 1 to 10 and differ from ACT 1 to        10 in one, two or three amino acids in positions other than        positions 60, 114, 121, 131, 135, 148, 153, 157, and 159.

Also considered are analogs of ACT1 (SEQ ID NO 1), in which

the amino acid in position 114 is A, E, N, R or S;

the amino acid in position 131 is N, S, T or V;

the amino acid in position 135 is D, N or T;

the amino acid in position 148 is D, E or Q;

the amino acid in position 157 is A, G, L, T, P or W; and

the amino acid in position 159 is E, F, M, R or S.

Likewise considered are analogs of ACT10 (SEQ ID NO 12), in which

the amino acid in position 60 is M or I;

the amino acid in position 114 is A, E, N, R or S;

the amino acid in position 121 is A or V;

the amino acid in position 131 is N, S, T or V;

the amino acid in position 135 is D, N or T;

the amino acid in position 148 is D, E, Q or V;

the amino acid in position 153 is L or S;

the amino acid in position 157 is A, G, L, T, P or W; and

the amino acid in position 159 is E, F, M, R, S or L.

Preferred are the proteins called alkylcytosine transferase (ACT) 1, 2,7, 8, and 10. Particularly preferred is the protein ACT10 with the aminoacid sequence shown in SEQ ID NO:12. Also preferred are homologs ofACT10 which differ thereof in one amino acid in positions other thanpositions 60, 114, 121, 131, 135, 148, 153, 157, and 159.

TABLE 1 Amino acid sequences of ^(N)AGT and ACT 1 to 10 Residue 60 114121 131 135 148 153 157 159 ^(N)AGT  M Y A K S V L G E SEQ ID NO: 2 ACT1M R A S D E L G M SEQ ID NO: 1 ACT2 M A A V D Q L W R ACT3 M S A T T D LP E ACT4 M N A N D E L A S ACT5 M A A T S E L K E ACT6 M E A R A E L E EACT7 M S A V D Q L L R ACT8 M E A N N E L T F ACT9 M E A N D V L P FACT10 I E V N D V S P L SEQ ID NO: 12

The invention further relates to a method for the production ofalkylcytosine transferases, characterized in that a DNA encoding AGT orACT is randomized by saturation mutagenesis in up to ten amino acidpositions, the obtained library transformed into suitable phagemids, thedesired phages selected by reaction with a benzylcytosine carrying alabel, and phages to which the label was transferred then isolated usingmagnetic beads covered with antibodies directed to the label. Theinvention also relates to the products of such method of directedevolution.

Saturation mutagenesis is well known in the art and is, for example,accomplished as described by Dube, D. K. and Loeb, L. A., Biochemistry,28:5703-5707, 1989.

Methods of directed evolution using phages and phagemids are also wellknown and are, for example, described in Smith, G. P. and Petrenko, V.A., Chem. Rev. 97:391-410, 1997; Hoess, R. H. et al., Chem. Rev.101:3205-3218, 2001. Preferred methods use phagemids pAK100 in a systemas described by Krebber, A., Bornhauser, S., Burmester, J., Honegger,A., Willuda, J., Bosshard, H. R. and Plückthun, A., J. Immunol. Methods201:35-55, 1997.

Compounds suitable for selection of the desired clones are thesubstrates as described under formula (I) and being subject to thisinvention, in particular substrates of formula (I) wherein R₁ ispara-substituted phenyl. The label of such a substrate being transferredto the desired ACT may be any label to which antibodies can easily beobtained, and is not restricted to a fluorescent dye label or any otherspectroscopic label.

Separation using magnetic beads carrying antibodies to a particularlabel are also well known in the art, and are described, for example, inGronemeyer et al., Protein Eng. Des. Sel. 19:309-316, 2006.

In a further aspect, the invention relates to compounds of formula (I)

wherein

R₁ is an aromatic or a heteroaromatic group, or an optionallysubstituted unsaturated alkyl, cycloalkyl or heterocyclyl group with thedouble bond connected to OCH₂—;

R₂ is a linker; and

L is a label or a plurality of same or different labels.

R₁ as an aromatic group is preferably phenyl or naphthyl, in particularphenyl, e.g. phenyl substituted by R₂ in para or meta position.

A heteroaromatic group R₁ is a mono- or bicyclic heteroaryl groupcomprising zero, one, two, three or four ring nitrogen atoms and zero orone oxygen atom and zero or one sulfur atom, with the proviso that atleast one ring atom is a nitrogen, oxygen or sulfur atom, and which has5 to 12, preferably 5 or 6 ring atoms; and which in addition to carryinga substituent R₂ may be unsubstituted or substituted by one or more,especially one, further substituents selected from the group consistingof lower alkyl, such as methyl, lower alkoxy, such as methoxy or ethoxy,halogen, e.g. chlorine, bromine or fluorine, halogenated lower alkyl,such as trifluoromethyl, or hydroxy.

Preferably the mono- or bicyclic heteroaryl group R₁ is selected from2H-pyrrolyl, pyrrolyl, imidazolyl, benzimidazolyl, pyrazolyl, indazolyl,purinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 4H-quinolizinyl,isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalyl,quinazolinyl, quinolinyl, pteridinyl, indolizinyl, 3H-indolyl, indolyl,isoindolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl,tetrazolyl, furazanyl, benzo[d]-pyrazolyl, thienyl, and furanyl. Morepreferably the mono-or bicyclic heteroaryl group is selected from thegroup consisting of pyrrolyl, imidazolyl, such as 1H-imidazol-1-yl,benzimidazolyl, such as 1-benzimidazolyl, indazolyl, especially5-indazolyl, pyridyl, e.g. 2-, 3- or 4-pyridyl, pyrimidinyl, especially2-pyrimidinyl, pyrazinyl, isoquinolinyl, especially 3-isoquinolinyl,quinolinyl, especially 4- or 8-quinolinyl, indolyl, especially3-indolyl, thiazolyl, triazolyl, tetrazolyl, benzo[d]pyrazolyl, thienyl,and furanyl.

In a particularly preferred embodiment of the invention the heteroarylgroup R₁ is thienyl, especially 2-thienyl, carrying the furthersubstituent R₂ in 3-, 4- or 5-position, preferably 4-position, or3-thienyl, carrying the further substituent R₂ in 4-position.

An optionally substituted unsaturated alkyl group R₁ is 1-alkenylcarrying the further substituent R₂ in 1- or 2-position, preferably in2-position, or 1-alkynyl. Substituents considered in 1-alkenyl are e.g.lower alkyl, e.g. methyl, lower alkoxy, e.g. methoxy, lower acyloxy,e.g. acetoxy, or halogenyl, e.g. chloro. In a particularly preferredembodiment of the invention R₁ is 1-alkynyl.

An optionally substituted unsaturated cycloalkyl group is a cycloalkenylgroup with 5 to 7 carbon atoms unsaturated in 1-position, e.g.1-cyclopentenyl or 1-cyclohexenyl, carrying the further substituent R₂in any position. Substituents considered are e.g. lower alkyl, e.g.methyl, lower alkoxy, e.g. methoxy, lower acyloxy, e.g. acetoxy, orhalogenyl, e.g. chloro.

An optionally substituted unsaturated heterocyclyl group has 3 to 12atoms, 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur, anda double bond in the position connecting the heterocyclyl group tomethylene in OCH₂—. Substituents considered are e.g. lower alkyl, e.g.methyl, lower alkoxy, e.g. methoxy, lower acyloxy, e.g. acetoxy, orhalogenyl, e.g. chloro.

In particular, an optionally substituted unsaturated heterocyclyl groupis a partially saturated heteroaromatic group as defined hereinbeforefor a heteroaromatic group R₁. An example of such a heterocyclyl groupis isoxazolidinyl, especially 3-isoxazolidinyl carrying the furthersubstituent in 5-position, or 5-isoxazolidinyl, carrying the furthersubstituent in 3-position.

A linker group R₂ is preferably a flexible linker connecting a label Lor a plurality of same or different labels L to the substrate. Linkerunits are chosen in the context of the envisioned application, i.e. inthe transfer of the substrate to a fusion protein comprising ACT. Theyalso increase the solubility of the substrate in the appropriatesolvent. The linkers used are chemically stable under the conditions ofthe actual application. The linker does not interfere with the reactionwith ACT nor with the detection of the label L, but may be constructedsuch as to be cleaved at some point in time after the reaction of thecompound of formula (I) with the fusion protein comprising ACT.

A linker R₂ is a straight or branched chain alkylene group with 1 to 300carbon atoms, wherein optionally

(a) one or more carbon atoms are replaced by oxygen, in particularwherein every third carbon atom is replaced by oxygen, e.g. apoylethyleneoxy group with 1 to 100 ethyleneoxy units;

(b) one or more carbon atoms are replaced by nitrogen carrying ahydrogen atom, and the adjacent carbon atoms are substituted by oxo,representing an amide function —NH—CO—;

(c) one or more carbon atoms are replaced by oxygen, and the adjacentcarbon atoms are substituted by oxo, representing an ester function—O—CO—;

(d) the bond between two adjacent carbon atoms is a double or a triplebond, representing a function —CH═CH— or —C≡C—;

(e) one or more carbon atoms are replaced by a phenylene, a saturated orunsaturated cycloalkylene, a saturated or unsaturated bicycloalkylene, abridging heteroaromatic or a bridging saturated or unsaturatedheterocyclyl group;

(f) two adjacent carbon atoms are replaced by a disulfide linkage —S—S—;or a combination of two or more, especially two or three, alkyleneand/or modified alkylene groups as defined under (a) to (f)hereinbefore, optionally containing substituents.

Substituents considered are e.g. lower alkyl, e.g. methyl, lower alkoxy,e.g. methoxy, lower acyloxy, e.g. acetoxy, or halogenyl, e.g. chloro.

Further substituents considered are e.g. those obtained when an α-aminoacid, in particular a naturally occurring a-amino acid, is incorporatedin the linker R₂ wherein carbon atoms are replaced by amide functions—NH—CO— as defined under (b).

In such a linker, part of the carbon chain of the alkylene group R₂ isreplaced by a group —(NH—CHR—CO)_(n)— wherein n is between 1 and 100 andR represents a varying residue of an α-amino acid.

A further substituent is one which leads to a photocleavable linker R₂,e.g. an o-nitrophenyl group. In particular this substituento-nitrophenyl is located at a carbon atom adjacent to an amide bond,e.g. in a group —NH—CO—CH₂—CH(o-nitrophenyl)-NH—CO—, or as a substituentin a polyethylene glycol chain, e.g. in a group—O—CH₂—CH(o-nitrophenyl)-O—. Other photocleavable linkers considered aree.g. phenacyl, alkoxybenzoin, benzylthioether and pivaloyl glycolderivatives.

A phenylene group replacing carbon atoms as defined under (e)hereinbefore is e.g. 1,2-, 1,3-, or preferably 1,4-phenylene. In aparticular embodiment, the phenylene group is further substituted by anitro group, and, combined with other replacements as mentioned aboveunder (a), (b), (c), (d), and (f), represents a photocleavable group,and is e.g. 4-nitro-1,3-phenylene, such as in—CO—NH—CH₂-4-nitro-1,3-phenylene-CH(CH₃)—O—CO—, or2-methoxy-5-nitro-1,4-phenylene, such as in—CH₂—O-2-methoxy-5-nitro-1,4-phenylene-CH(CH₃)—O—. Other particularembodiments representing photocleavable linkers are e.g.-1,4-phenylene-CO—CH₂—O—CO—CH₂— (a phenacyl group),-1,4-phenylene-CH(OR)—CO-1,4-phenylene- (an alkoxybenzoin), or-3,5-dimethoxy-1,4-phenylene-CH₂—O— (a dimethoxybenzyl moiety). Asaturated or unsaturated cycloalkylene group replacing carbon atoms asdefined under (e) hereinbefore is derived from cycloalkyl with 3 to 7carbon atoms, preferably from cyclopentyl or cyclohexyl, and is e.g.1,2- or 1,3-cyclopentylene, 1,2-, 1,3-, or preferably 1,4-cyclohexylene,or also 1,4-cyclohexylene being unsaturated e.g. in 1- or in 2-position.A saturated or unsaturated bicycloalkylene group replacing carbon atomsas defined under (e) hereinbefore is derived from bicycloalkyl with 7 or8 carbon atoms, and is e.g. bicyclo[2.2.1] heptylene orbicyclo[2.2.2]octylene, preferably 1,4-bicyclo[2.2.1]heptyleneoptionally unsaturated in 2-position or doubly unsaturated in 2- and5-position, and 1,4-bicyclo[2.2.2]octylene optionally unsaturated in2-position or doubly unsaturated in 2- and 5-position. A bridgingheteroaromatic group replacing carbon atoms as defined under (e)hereinbefore is e.g. triazolidene, preferably 1,4-triazolidene, orisoxazolidene, preferably 3,5-isoxazolidene. A bridging saturated orunsaturated heterocyclyl group replacing carbon atoms as defined under(e) hereinbefore is e.g. derived from an unsaturated heterocyclyl groupas defined under R₁ above, e.g. isoxazolidinene, preferably3,5-isoxazolidinene, or a fully saturated heterocyclyl group with 3 to12 atoms, 1 to 3 of which are heteroatoms selected from nitrogen, oxygenand sulfur, e.g. pyrrolidinediyl, piperidinediyl, tetrahydrofuranediyl,dioxanediyl, morpholinediyl or tetrahydrothiophenediyl, preferably2,5-tetrahydrofuranediyl or 2,5-dioxanediyl. A particular heterocyclylgroup considered is a saccharide moiety, e.g. an α- or β-furanosyl or α-or β-pyranosyl moiety.

Cyclic substructures in a linker R₂ reduce the molecular flexibility asmeasured by the number of rotatable bonds within R₂, which leads to abetter membrane permeation rate, important for all in vivo labellingapplications.

A linker R₂ is preferably a straight chain alkylene group with 1 to 25carbon atoms or a straight chain polyethylene glycol group with 4 to 100ethyleneoxy units, optionally attached to the group R₁ by a —CH═CH— or—C≡C— group. Further preferred is a straight chain alkylene group with 1to 25 carbon atoms wherein carbon atoms are optionally replaced by anamide function —NH—CO—, and optionally carrying a photocleavablesubunit, e.g. o-nitrophenyl. Further preferred are branched linkerscomprising a polyethylene glycol group of 3 to 6 ethylene glycol unitsand alkylene groups wherein carbon atoms are replaced by amide bonds,and further carrying substituted amino and hydroxy functions. Otherpreferred branched linkers have dendritic (tree-like) structures whereinamine, carboxamide and/or ether functions replace carbon atoms of analkylene group.

A particularly preferred linker R₂ is a straight chain alkylene group of2 to 20 carbon atoms wherein one or two carbon atoms are replaced bynitrogen and which is optionally substituted by oxo adjacent to thenitrogen.

Another particularly preferred linker R₂ is a straight chain alkylenegroup of 10 to 40 carbon atoms optionally substituted by oxo wherein 3to 12 carbon atoms are replaced by oxygen and one or two carbon atomsare replaced by nitrogen, optionally substituted by oxo adjacent tonitrogen.

A linker R₂ may carry one or more same or different labels, e.g. 1 to100 same or different labels, in particular 1 to 5, preferably one, twoor three, in particular one or two same or different labels.

Lower alkyl is alkyl with 1 to 7, preferably from 1 to 4 C atoms, and islinear or branched; preferably, lower alkyl is butyl, such as n-butyl,sec-butyl, isobutyl, tert-butyl, propyl, such as n-propyl or isopropyl,ethyl or methyl. Most preferably, lower alkyl is methyl.

In lower alkoxy, the lower alkyl group is as defined hereinbefore. Loweralkoxy denotes preferably n-butoxy, tert-butoxy, iso-propoxy, ethoxy, ormethoxy, in particular methoxy.

In lower acyloxy, lower acyl has the meaning of formyl or loweralkylcarbonyl wherein lower alkyl is defined as hereinbefore. Loweracyloxy denotes preferably n-butyroxy, n-propionoxy, iso-propionoxy,acetoxy, or formyloxy, in particular acetoxy.

Halogen is fluoro, chloro, bromo or iodo, in particular chloro.

The label L of the substrate can be chosen by those skilled in the artdependent on the application for which the fusion protein is intended.Labels are such that the labelled fusion protein carrying label L iseasily detected or separated from its environment. Other labelsconsidered are those which are capable of sensing and inducing changesin the environment of the labelled fusion protein and/or the substrate,or labels which aid in manipulating the fusion protein by the physicaland/or chemical properties of the substrate and specifically introducedinto the fusion protein. A label as understood in the context of theinvention is a substituent different from hydrogen or from standardfunctional groups, in particular different from hydrogen, hydroxy,amino, halogen, carboxylate, carboxamide, carboxylic ester, nitrile,cyanate, isocyanate, sulfonate, sulfonamide, sulfonic ester, aldehyde,ketone, ether, and thioether substituent.

Examples of a label include a spectroscopic probe such as a fluorophoreor a chromophore, a magnetic probe or a contrast reagent; aradioactively labelled molecule; a molecule which is one part of aspecific binding pair which is capable of specifically binding to apartner; a molecule that is suspected to interact with otherbiomolecules; a library of molecules that are suspected to interact withother biomolecules; a molecule which is capable of crosslinking to othermolecules; a molecule which is capable of generating hydroxyl radicalsupon exposure to H₂O₂ and ascorbate, such as a tethered metal-chelate; amolecule which is capable of generating reactive radicals uponirradiation with light, such as malachite green; a molecule covalentlyattached to a solid support, where the support may be a glass slide, amicrotiter plate or any polymer known to those proficient in the art; anucleic acid or a derivative thereof capable of undergoing base-pairingwith its complementary strand; a lipid or other hydrophobic moleculewith membrane-inserting properties; a biomolecule with desirableenzymatic, chemical or physical properties; or a molecule possessing acombination of any of the properties listed above.

Further labels L are positively charged linear or branched polymerswhich are known to facilitate the transfer of attached molecules overthe plasma membrane of living cells. This is of particular importancefor substances which otherwise have a low cell membrane permeability orare in effect impermeable for the cell membrane of living cells. A noncell permeable ACT substrate will become cell membrane permeable uponconjugation to such a group L. Such cell membrane transport enhancergroups L comprise, for example, a linear poly(arginine) of D- and/orL-arginine with 6-15 arginine residues, linear polymers of 6-15 subunitswhich each carry a guanidinium group, oligomers or short-length polymersof from 6 to up to 50 subunits, a portion of which have attachedguanidinium groups, and/or parts of the sequence of the HIV-tat protein,in particular the subunit Tat49-Tat57 (RKKRRQRRR in the one letter aminoacid code). The ACT substrate is covalently linked to this group Lthrough a linker R₂ as defined hereinbefore, which is preferably labileinside a living cell and may be degraded, e.g. by cleavage of an estergroup R₂ by intracellular esterases, leading directly or in a furtherreaction provoked by the cleavage of the ester function to a separationof the ACT substrate and the unit L enhancing cell membranepermeability.

Preferred as labels L are spectroscopic probes, and molecules which areone part of a specific binding pair which is capable of specificallybinding to a partner, so-called affinity labels. Also preferred aslabels L are molecules covalently attached to a solid support. Preferredspectroscopic probes are fluorophores.

When the label L is a fluorophore, a chromophore, a magnetic label, aradioactive label or the like, detection is by standard means adapted tothe label and whether the method is used in vitro or in vivo. If L is afluorophore the method can be compared to the applications of the greenfluorescent protein (GFP) which is genetically fused to a protein ofinterest and allows protein investigation in the living cell. Particularexamples of labels L are also boron compounds displaying non-linearoptical properties. Particularly preferred are labels such that L of onesubstrate (L₁) is one member and L of another substrate (L₂) is theother member of two interacting spectroscopic probes L₁/L₂, whereinenergy can be transferred nonradiatively between the donor and acceptor(quencher) when they are in close proximity (less than 10 nanometerdistance) through either dynamic or static quenching. Such a pair oflabels L₁/L₂ changes its spectroscopic properties on reaction of thelabelled substrate carrying L₁ with the ACT fusion protein and anothertype of labelled substrate (e.g. a benzylguanine type) carrying L₂ withanother corresponding fusion protein, e.g. an AGT fusion protein. Anexample of such a pair of labels L₁/L₂ is a FRET pair explained below inmore detail.

Particular fluorophores considered are: Alexa Fluor dyes, includingAlexa Fluor 350, 488, 532, 546, 555, 635 and 647 (Invitrogen Corp.,Carlsbad, Calif. 92008, USA, see also Panchuk-Voloshina, N. et al., J.Histochem. & Cytochem. 47:1179-1188, 1999); coumarins such as7-dimethylamino-coumarin-4-acetic acid (succinimidyl ester supplied asproduct D374 by Invitrogen Molecular Probes),7-amino-4-methyl-coumarin-3-acetic acid and7-diethylamino-coumarin-3-carboxylic acid; Cyanine-3 (Cy 3), Cyanine 5(Cy 5) and Cyanine 5.5 (Cy 5.5) (Amersham—GE Healthcare, Solingen,Germany); ATTO 488, ATTO 532, ATTO 600 and ATTO 655 (Atto-Tec, D57076Siegen, Germany); DY-505, DY-547, DY-632 and DY-647 (Dyomics, Jena,Germany); and 5(6)-carboxyfluorescein anddifluoro-5(6)-carboxyfluorescein (Oregon Green). These particular labelson the substrate of formula (I) may be combined with the knownAGT—benzylguanine system wherein the label on the benzylguanine is aquencher to create a FRET pair with the fluorophore on the substrate offormula (I) reacting with ACT. Such quenchers are: QSY 35, QSY 9 and QSY21 (Invitrogen Molecular Probes); BHQ-1, BHQ-2 and BHQ-3 (Black HoleQuencher™ of Biosearch Technologies, Inc., Novato, Calif. 94949, USA);ATTO 540Q and ATTO 612Q (Atto-Tec, D57076 Siegen, Germany);4-dimethylamino-azobenzene-4′-sulfonyl derivatives (Dabsyl) and4-dimethylaminoazobenzene-4′-carbonyl derivatives (Dabcyl).

Depending on the properties of the label L, the fusion proteincomprising protein of interest and ACT may be bound to a solid supporton reaction with the substrate. The label L of the substrate reactingwith the fusion protein comprising ACT may already be attached to asolid support when entering into reaction with ACT, or may subsequently,i.e. after transfer to ACT, be used to attach the labelled ACT fusionprotein to a solid support. The label may be one member of a specificbinding pair, the other member of which is attached or attachable to thesolid support, either covalently or by any other means. A specificbinding pair considered is e.g. biotin and avidin or streptavidin.Either member of the binding pair may be the label L of the substrate,the other being attached to the solid support. Further examples oflabels allowing convenient binding to a solid support are e.g. maltosebinding protein, glycoproteins, FLAG tags, or reactive substituentsallowing chemoselective reaction between such substituent with acomplementary functional group on the surface of the solid support.Examples of such pairs of reactive substituents and complementaryfunctional group are e.g. amine and activated carboxy group forming anamide, azide and a propiolic acid derivative undergoing a 1,3-dipolarcycloaddition reaction, amine and another amine functional groupreacting with an added bifunctional linker reagent of the type ofactivated bis-dicarboxylic acid derivative giving rise to two amidebonds, or other combinations known in the art.

Examples of a convenient solid support are e.g. glass surfaces such asglass slides, microtiter plates, and suitable sensor elements, inparticular functionalized polymers (e.g. in the form of beads),chemically modified oxidic surfaces, e.g. silicon dioxide, tantalumpentoxide or titanium dioxide, or also chemically modified metalsurfaces, e.g. noble metal surfaces such as gold or silver surfaces.Irreversibly attaching and/or spotting ACT substrates may then be usedto attach ACT fusion proteins in a spatially resolved manner,particularly through spotting, on the solid support representing proteinmicroarrays, DNA microarrays or arrays of small molecules.

When the label L is capable of generating reactive radicals, such ashydroxyl radicals, upon exposure to an external stimulus, the generatedradicals can then inactivate the ACT fusion proteins as well as thoseproteins that are in close proximity of the ACT fusion protein, allowingto study the role of these proteins. Examples of such labels aretethered metal-chelate complexes that produce hydroxyl radicals uponexposure to H₂O₂ and ascorbate, and chromophores such as malachite greenthat produce hydroxyl radicals upon laser irradiation. The use ofchromophores and lasers to generate hydroxyl radicals is also known inthe art as chromophore assisted laser induced inactivation (CALI). Inthe present invention, labelling ACT fusion proteins with substratescarrying chromophores as label L, such as malachite green, andsubsequent laser irradiation inactivates the labelled ACT fusion proteinas well as those proteins that interact with the ACT fusion protein in atime-controlled and spatially-resolved manner. This method can beapplied both in vivo or in vitro. Furthermore, proteins which are inclose proximity of the ACT fusion protein can be identified as such byeither detecting fragments of that protein by a specific antibody, bythe disappearance of those proteins on a high-resolution2D-electrophoresis gels or by identification of the cleaved proteinfragments via separation and sequencing techniques such as massspectrometry or protein sequencing by N-terminal degradation.

When the label L is a molecule that can cross-link to other proteins,e.g. a molecule containing functional groups such as maleimides, activeesters or azides and others known to those proficient in the art,contacting such labelled ACT substrates with ACT fusion proteins thatinteract with other proteins (in vivo or in vitro) leads to the covalentcross-linking of the ACT fusion protein with its interacting protein viathe label. This allows the identification of the protein interactingwith the ACT fusion protein. Labels L for photo cross-linking are e.g.benzophenones. In a special aspect of cross-linking the label L is amolecule which is itself an ACT substrate leading to dimerization of theACT fusion protein. The chemical structure of such dimers may be eithersymmetrical (homodimers) or unsymmetrical (heterodimers).

Other labels L considered are for example fullerenes, boranes forneutron capture treatment, nucleotides or oligonucleotides, e.g. forself-addressing chips, peptide nucleic acids, and metal chelates, e.g.platinum chelates that bind specifically to DNA.

A particular biomolecule with desirable enzymatic, chemical or physicalproperties is methotrexate. Methotrexate is a tight-binding inhibitor ofthe enzyme dihydrofolate reductase (DHFR). Compounds of formula (I)wherein L is methotrexate belong to the well known class of so-called“chemical inducers of dimerization” (CIDs). Using fusion proteins of ACTwith the DNA-binding domain LexA, and adding DHFR with thetranscriptional activation domain B42 to the in vivo labeling of the ACTfusion protein with a compound of formula (I) wherein L is methotrexateinduces the coupling (“dimerization”) of the ACT-LexA fusion protein andDHFR-B42 fusion protein, leading to spatial proximity of LexA and B42and subsequent stimulation of transcription.

If the substrate carries two or more labels, these labels may beidentical or different.

Particular preferred combinations are two different affinity labels, orone affinity label and one chromophore label, in particular one affinitylabel and one fluorophore label, or a pair of spectroscopic interactinglabels L₁/L₂, e.g. a FRET pair.

Preferred are compounds of formula (I) wherein R₁ is phenyl, inparticular para-substituted phenyl.

Preferred are compounds of formula (I) wherein L is a spectroscopicprobe, e.g. a fluorophore. Likewise preferred are compounds wherein L isa molecule representing one part of a specific binding pair, andcompounds wherein L is a molecule covalently attached to a solidsupport.

Most preferred are the compounds of the Examples.

The invention further relates to a method for detecting and/ormanipulating a protein of interest, wherein the protein of interest isincorporated into an ACT fusion protein, the ACT fusion protein iscontacted with a compound of formula (I) carrying a label as describedhereinbefore, and the ACT fusion protein is detected and optionallyfurther manipulated using the label in a system designed for recognisingand/or handling the label.

In the method of the present invention a protein or peptide of interestis fused to an ACT. The protein or peptide of interest may be of anylength and both with and without secondary, tertiary or quaternarystructure, and preferably consists of at least twelve amino acids and upto 2000 amino acids. Examples of such protein or peptide of interest aree.g. enzymes, DNA-binding proteins, transcription regulating proteins,membrane proteins, nuclear receptor proteins, nuclear localizationsignal proteins, protein cofactors, small monomeric GTPases, ATP-bindingcassette proteins, intracellular structural proteins, proteins withsequences responsible for targeting proteins to particular cellularcompartments, proteins generally used as labels or affinity tags, anddomains or subdomains of the aforementioned proteins. The protein orpeptide of interest is preferably fused to ACT by way of a linker whichmay be cleaved by an enzyme, e.g. at the DNA stage by suitablerestriction enzymes and/or linkers cleavable by suitable enzymes at theprotein stage.

The ACT has the property of transferring a label present on a substrate,i.e. on the compound of formula (I), to one of the cysteine residues ofthe ACT forming part of a fusion protein. In preferred embodiments, theACT is selected from the group consisting of ACT 1 to ACT 10 as definedhereinbefore, and homologs thereof. Particularly preferred is ACT 10.

The fusion protein comprising protein of interest and an ACT iscontacted with a particular substrate of formula (I). Conditions ofreaction are selected such that the ACT reacts with the substrate andtransfers the label of the substrate. Usual conditions are a buffersolution at around pH 7 at room temperature, e.g. around 25° C. However,it is understood that ACT reacts also under a variety of otherconditions, and those conditions mentioned here are not limiting thescope of the invention.

The label L of the substrate is chosen by those skilled in the artdependent on the application for which the fusion protein is intended.After contacting the fusion protein comprising ACT with the substrate,the label L is covalently bonded to the fusion protein. The labelled ACTfusion protein is then further manipulated and/or detected by virtue ofthe transferred label. The label L may consist of a plurality of same ordifferent labels. If the substrate contains more than one label L, thecorresponding labelled ACT fusion protein will also comprise more thanone label which gives more options for further manipulating and/ordetecting the labelled fusion protein.

“Detected” in the sense of the present invention means that the fusionprotein with ACT carrying label L can be localised due to the propertiesof the label, and the amount of fusion protein determined eitherdirectly or by reference to a standard. “Manipulated” in the sense ofthe present invention means that the fusion protein with ACT carryinglabel L can be reacted further due to the properties of the label, e.g.isolated from the in vitro or in vivo system, enriched and purified,i.e. separated from other proteins and/or non-proteinaceous material,brought in solution (e.g. especially if the fusion protein is notsoluble), precipitated (e.g. especially if the fusion protein issoluble) or otherwise fixed to a solid, and also further treated e.g. bycleaving the linker which splits off the label and regenerates thefusion protein. The skilled person well understands the manypossibilities for further handling the fusion protein carrying label Ldue to the properties of the label L and of the linker R₂ connecting Lto the fusion protein.

In vitro, the reaction of the ACT fusion protein with the substrate ofthe invention can generally be either performed in cell extracts or withpurified or enriched forms of the ACT fusion protein.

If experiments with the substrates of the present invention are done invivo or in cell extracts, the reaction of the endogenous AGT will notdisturb the reaction of an ACT fusion protein with a substrate of theformula (I), since said substrate does not (or at least not detectably)interact with endogenous AGT, only with ACT. This is a substantialadvantage of the present combination ACT-substrate of formula (I) overthe standard combination of AGT-benzylguanine type substrate describedin the prior art.

Particular fluorophores labels on the substrate of formula (I) asdescribed hereinbefore may be combined with the known AGT-benzylguaninesystem wherein the label on the benzylguanine is a quencher to create aFRET pair with the fluorophore on the substrate of formula (I) reactingwith ACT. Preferred are the quenchers listed hereinbefore as labels Lfor substrates of formula (I).

Alternatively, the label L on the substrate of formula (I) may be one ofthe quenchers mentioned above, and the reaction with ACT accomplished ina mixture with a AGT fusion protein-benzylguanine substrate combinationwherein the label on benzylguanine is one of the mentioned fluorophores.

Substrates of the invention are generally prepared by standard methodsknown in the art. A useful starting material is2-chloropyrimidin-4-amine or another cytosine derivative with anactivated leaving group at position 2 of the pyrimidine ring. Thiscompound is then reacted with an alcohol of formula HO—CH₂—R₁—R₂-L or ananalogous compound HO—CH₂—R₁—R₂′ wherein R₂′ is precursor of a linker R₂allowing the introduction of a label L and thereby completing theresidue R₂-L. Particular useful precursor R₂′ are those with an aminofunction, which allows a condensation reaction with labels carrying acarboxyl function, e.g. in the form of a succinimidyl ester giving riseto linker R₂ incorporating an amide function.

Appropriate protecting groups for the envisioned functionalities can bechosen by those skilled in the art, and are e.g. summarized in Greene,T. W. and Wuts, P. G. M. in “Protective Groups in Organic Synthesis”,John Wiley & Sons, New York 1991.

The invention further relates to methods of transferring a label fromthese substrates of formula (I) to alkylcytosine transferases (ACTs) andACT fusion proteins.

The combination ACT-substrate of formula (I) is particularly suitablefor tracing two different proteins in combination with the knownAGT-benzylguanine type substrate, since the substrate of formula (I) andbenzylguanine type substrates transfer their labels selectively to ACTand AGT fusion proteins, respectively.

In order to demonstrate the feasibility of specific dual labelling usingACTs, an equimolar mixture of hexahistidine tagged ^(N)AGT(6×His-^(N)AGT) and GST-ACT1 fusion protein was incubated with eitherBGFL, BGCy5, BCFL, BCCy5 or an equimolar mixture of BGCy5/BCFL orBGFL/BCCy5. Incubation with BGFL or BGCy5 led to a complete labelling of6×His-^(N)AGT and very low labelling of GST-ACT1 (estimated to be lessthan 5% in both case). In an opposite way, incubation with BCFL or BCCy5led to a complete labelling of GST-ACT1 and a partial labelling of6×His-^(N)AGT (76% and 6%, respectively). However, incubation with anequimolar mixture of BGCy5/BCFL (or BGFL/BCCy5) led to specific dualcolour labelling, i.e. specific labelling of 6×His-^(N)AGT with Cy5 dye(or fluorescein, respectively) and specific labelling of GST-ACT1 withfluorescein (or Cy5 dye, respectively). In both case, cross labellinghad been evaluated to be less than 1%.

TABLE 2 Activity of ^(N)AGT and ACT 1 to 10 as GST-fusion protein withBCFL and BGFL [s⁻¹ · M⁻¹] k_(BCFL) k_(BGFL) ^(N)AGT 26 28000 ACT 1 730<10 ACT 2 330 <10 ACT 3 100 n.d. ACT 4 <100 n.d. ACT 5 350 210 ACT 6 460160 ACT 7 260 <10 ACT 8 600 <10 ACT 9 90 <1 ACT 10 1130 10 n.d.: notdetermined

EXAMPLES

Abbreviations:

BC=Benzylcytosine

BG=Benzylguanine

DTT=Dithiothreitol

DMF=Dimethylformamide

MPLC=Medium pressure liquid chromatography

PBS=Phosphate buffered saline

RT=Room temperature

TFA=Trifluoroacetic acid

TEA=Triethylamine

Example 1N-(4-((4-Aminopyrimidin-2-yloxy)methyl)benzyl)-2,2,2-trifluoroacetamide(1)

760 mg (3.25 mmol) 2,2,2-Trifluoro-N-(4-hydroxymethyl-benzyl)-acetamideis dissolved in 3 mL dry dimethylacetamide under argon atmosphere, and273 mg (8.15 mmol) NaH is added over 5 min. 211 mg (1.63 mmol)2-Chloropyrimidin-4-amine is then added and the solution stirred at 90°C. over night. 1 mL Water is added carefully to quench all excess NaH,and the mixture poured into 50 ml of 0.5 N HCl. The crude product isextracted with ethyl acetate, the combined organic phases washed withbrine and dried over MgSO₄. After evaporation of the solvent, theproduct is purified by flash column chromatography (gradient ethylacetate:cyclohexane from 1:1 to 3:1). Yield: 350 mg (52%). ESI-MS m/z327 [M+H]⁺.

Example 2 2-(4-(Aminomethyl)benzyloxy)4-aminopyrimidine (2)

150 mg (0.46 mmol) ofN-(4-((4-Aminopyrimidin-2-yloxy)methyl)benzyl)-2,2,2-trifluoroacetamide(1) is dissolved in 2 mL methanol and treated with 5 mL methylamine (33%in ethanol). The reaction mixture is stirred at room temperature overnight and all volatiles are removed in vacuo. The product is usedwithout further purification in the next step. ESI-MS m/z 231 [M+H]⁺.

Example 3N-[4-(4-Aminopyrimidin-2-yloxymethyl)-benzyl]-tetramethylrhodamine-6-carboxamide(3) andN-[4-(4-Aminopyrimidin-2-yloxymethyl)-benzyl]-tetramethylrhodamine-5-carboxamide(4)

2-(4-(Aminomethyl)benzyloxy)pyrimidin-4-amine (2) (3.6 mg, 0.016 mmol)and 5(6)-carboxytetramethylrhodamine succinimidyl ester (8.2 mg, 0.016mmol) are dissolved in 800 μL DMF with 2.4 μL TEA and heated overnightat 31° C. The solvent is evaporated in vacuo and the compounds areisolated by reversed phase MPLC (medium pressure liquid chromatography)on a C18 column using a linear gradient of water:acetonitrile (from 95:5to 20:80 in 20 min, 0.08% TFA). MS (ESI) m/z 643 [M−Cl]⁺.

Example 4N-[4-(4-Aminopyrimidin-2-yloxymethyl)-benzyl]-fluorescein-6-carboxamide(5), BCFL

2-(4-(Aminomethyl)benzyloxy)pyrimidin-4-amine (2) (3.9 mg, 0.017 mmol)and 5(6)-carboxyfluorescein succinimidyl ester (8.3 mg, 0.017 mmol) aredissolved in 800 μL DMF with 2.6 μL TEA and heated overnight at 31° C.The solvent is evaporated in vacuo and the product is isolated byreversed phase MPLC on a C18 column using a linear gradient ofwater:acetonitrile (from 95:5 to 20:80 in 20 min, 0.08% TFA). MS (ESI)m/z 589 [M+H]⁺. Depending on the purification method, the product mayalso contain the corresponding isomer 5-carboxamide.

Example 5N-[4-(4-Aminopyrimidin-2-yloxymethyl)-benzyl]-diacetylfluorescein-6-carboxamide(6) andN-[4-(4-Aminopyrimidin-2-yloxymethyl)-benzyl]-diacetylfluorescein-5-carboxamide(7)

2-(4-(Aminomethyl)benzyloxy)pyrimidin-4-amine (2) (4.1 mg, 0.018 mmol)and 5(6)-carboxyfluorescein diacetate succinimidyl ester (10 mg, 0.018mmol) are dissolved in 800 μL DMF with 2.7 μL TEA and heated overnightat 31° C. The solvent is evaporated under vacuum and the compoundsisolated by reversed phase MPLC on a C18 column using a linear gradientof water:acetonitrile (from 95:5 to 20:80 in 20 min, 0.08% TFA). MS(ESI) m/z 673 [M+H]⁺.

Example 6N-[4-(4-Aminopyrimidin-2-yloxymethyl)-benzyl]-DY647-carboxamide (8)

2-(4-(Aminomethyl)benzyloxy)pyrimidin-4-amine (2) (1.5 mg, 0.007 mmol)and DY-547-NHS (Dyomics dye) (5 mg, 0.007 mmol) are dissolved in 500 μLDMF with 1.0 μL TEA and heated overnight at 31° C. The solvent isevaporated under vacuum and the product is isolated by reversed phaseMPLC on a C18 column using a linear gradient of water:acetonitrile (from95:5 to 20:80 in 20 min, 0.08% TFA). MS (ESI) m/z 853 [M−Na]⁻.

Example 7N-[4-(4-Aminopyrimidin-2-yloxymethyl)-benzyl]-DY547-carboxamide (9)

2-(4-(Aminomethyl)benzyloxy)pyrimidin-4-amine (2) (1.5 mg, 0.007 mmol)and DY-547-NHS (Dyomics dye) (5 mg, 0.007 mmol) are dissolved in 500 μLDMF with 1.0 μL TEA and heated overnight at 31° C. The solvent isevaporated under vacuum and the product is isolated by reversed phaseMPLC on a C18 column using a linear gradient of water:acetonitrile (from95:5 to 20:80 in 20 min, 0.08% TFA). MS (ESI) m/z 827 [M−Na]⁻.

Example 8 N-[4-(4-Aminopyrimidin-2-yloxymethyl)-benzyl]-Cy5-carboxamide(10) BCCy5

2-(4-(Aminomethyl)benzyloxy)pyrimidin-4-amine (2) (1.5 mg, 0.007 mmol)and Cy5-NHS (GE healthcare dye) (5.4 mg, 0.007 mmol) are dissolved in500 μL DMF with 1.0 μL TEA and heated overnight at 31° C. The solvent isevaporated under vacuum and the product is isolated by reversed phaseMPLC on a C18 column using a linear gradient of water:acetonitrile (from95:5 to 20:80 in 20 min, 0.08% TFA). MS (ESI) m/z 868 [M−Na]⁻.

Example 9 O⁶-[4-(Cy5)-Aminomethyl]-benzyl guanine (11), BGCy5

O⁶-4-aminomethyl-benzyl-guanine (1.9 mg, 0.007 mmol) and Cy5-NHS (GEhealthcare dye) (5.4 mg, 0.007 mmol) are dissolved in 500 μL DMF with1.0 μL TEA and heated overnight at 31° C. The solvent is evaporatedunder vacuum and the product is isolated by reversed phase MPLC on a C18column using a linear gradient of water:acetonitrile (from 95:5 to 20:80in 20 min, 0.08% TFA). MS (ESI) m/z 908 [M−Na]⁻.

Example 10

The following compounds are prepared from2-(4-(aminomethyl)benzyloxy)pyrimidin-4-amine (2) in analogy to Example3:

BC-Biotin of formula 12:

BC-PEG-Biotin of formula 13:

BC-360 of formula 14:

BC-430 of formula 15:

BC-Oregon Green consisting of a mixture of compounds of formula 16:

BC-Oregon Green dipivaloyl ester consisting of a mixture of compounds offormula 17:

BC-505 consisting of a mixture of compounds of formula 18:

Example 11 Library Construction and Phage Selection

Overlap extension PCR using primers 1-5 and ^(N)AGT gene (SEQ ID NO:2)as template allowed to randomize the residues 114, 131, 135, 148, 156,157 and 159 of ^(N)AGT. ^(N)AGT is a 182 amino acid mutant of ^(wt)AGTin which the last 25 amino acids are deleted and that possesses themutations K32I, L33F, C62A, Q115S, Q116H, K125A, A127T, R128A, G131K,G132T, M134L, R135S, N150Q, I151G, N152D, G153L, A154D, N157G and S159E(Gronemeyer et al., Protein Eng. Des. Set. 19:309-316, 2006). Primers 1(N, AGT, SEQ ID NO:3) and 2 (C, AGT, SEQ ID NO:4) contain Sfi1restriction sites; Primer 3 (SEQ ID NO:5) contains the randomized basesfor randomization at position 114; Primer 4 (SEQ ID NO:6) contains therandomized bases for randomization at position 131 and 135; Primer 5(SEQ ID NO:7) contains the randomized bases for randomization atposition 148, 156, 157 and 159. The PCR product was ligated into phagedisplay vector pAK100 and the resulting construct was electroporatedinto E. coli XL1-blue (Stratagene, USA). This led to a librarycontaining about 10⁷ clones. Library cells were grown in 2YT medium (25μg/mL chloramphenicol, 1% glucose, 1 mM MgCl₂) at 37° C. until theoptical density OD₆₀₀ reached 0.6. Then 2×10¹⁰ VCS M13 helper phageswere added and the culture was incubated 30 min at 37° C. withoutshaking and 3 h at 37° C. at 170 rpm. Cells were harvested bycentrifugation (4000 rpm, 5 min, RT), resuspended in SB-MOPS (50 mM3-morpholinopropansulfonic acid, 25 μg/mL chloramphenicol, 70 μg/mLkanamycin, 1 mM MgCl₂) and incubated 1-4 h at 37° C. at 220 rpm, thenovernight at 24° C. at 220 rpm. Cells were pelleted and the supernatantcontaining phages adjusted to 1 mM DTT and stored at 4° C. prior toselections. For selection, BCFL (compound 5) was added to the phagesolution (1 mL) to a final concentration of 5 μM, gently rotated for 30min at room temperature. The reaction was quenched by addition of 8 μMBC and 200 μM BG. Phages were precipitated at 4° C. using polyethyleneglycol 8000 (4% w/v) and NaCl (3% w/v), centrifuged in a desktopcentrifuge (13000 rpm, 4° C.) and resuspended in 500 μL PBS. To thissolution, 500 μL of PBSMM (PBS with 4% skimmed milk powder) were addedand the solution was gently rotated for 60 min at room temperature. 200μL of magnetic beads covered with anti-fluorescein antibody (washedtwice with PBS and blocked for 60 min with PBSMM) were added to thephage preparation and rotated at 4° C. for 30 min. After immobilizationof labelled phages, the beads were washed 3 times with PBSMM, 5 timeswith PBST (PBS with 0.05% Tween-20), twice with PBS. Phages were elutedby incubation of the beads with 100 μL 0.1 M glycine, pH 2.5 for 5 minand the solution was neutralized with 50 μL 1 M Tris-HCl pH 8. E. coliJM101 were infected with eluted phages, plated on 2YT platessupplemented with 1% glucose and 25 μg/mL chloramphenicol, thenincubated overnight at 37° C. The next day, colonies were scraped offthe plates, aliquoted and stored at −80° C. prior to the next round ofselection. Six rounds of selection were performed.

Example 12 Characterization of Selected ACTs as GST Fusion Proteins

The genes of mutants isolated after phage selection according to Example9 were amplified by PCR using primers 6 (N, AGT, SEQ ID NO:8) and 7 (C,AGT, SEQ ID NO:9) that contain, respectively, BamH1 and EcoR1restriction sites for subsequent subcloning into pGEX-2T vector(Amersham Biosciences, Otelfingen, Switzerland). Expression andpurification of proteins as GST-ACT fusion proteins were performed aspreviously described for AGT fusion proteins (A. Juillerat, T.Gronemeyer, A. Keppler, S. Grendreizig, H. Pick, H. Vogel, K. Johnsson,Chem. Biol. 10:313, 2003). Reaction rates of labelling with BCFL(compound 5) and BG carrying fluorescein (BGFL, WO 02/08397) weredetermined by incubation of corresponding ACT protein (0.2-0.4 μM) withthe appropriate fluorogenic substrate (2-20 μM) in reaction buffer (50mM HEPES, pH 7.2, 1 mM DTT, 200 μg/mL of BSA) at 24° C. Samples weretaken at different times and the labelling reaction was quenched byaddition of 4× SDS buffer (8% SDS, 10% β-mercaptoethanol, 240 mM Tris pH6.8, 40% glycerol) and incubation at 95° C. for 5 min. The reactionadvancement was determined by detection of the fluorescent dye-labelledproteins in SDS-PAGE gel and quantification of fluorescence intensityusing a Pharox FX™ molecular imager. The data were fitted to apseudo-first order reaction model. Second-order rate constants were thenobtained by dividing the pseudo first-order constant by theconcentration of fluorogenic substrate.

Example 13 In vitro Specific Dual Labelling Assays

In vitro dual labelling assays were performed by incubating an equimolarmixture of GST-ACT1 and 6×His-^(N)AGT (0.5 μM final concentration) witheither BGFL (benzylguanine carrying fluorescein, WO 02/08397), BGCy5(benzylguanine carrying fluorescent dye Cy5, compound 11, Example 9),BCFL (compound 5, Example 4), BCCy5 (compound 10, Example 8) (5 μM) oran equimolar mixture of BGCy5/BCFL or BGFL/BCCy5 (5 μM each) in reactionbuffer (50 mM HEPES, pH 7.2, 1mM DTT, 200 μg/mL of BSA) at 24° C. for 60minutes. Labelling reactions were quenched by addition of 4× SDS buffer(8% SDS, 10% β-mercaptoethanol, 240 mM Tris pH 6.8, 40% glycerol) andincubation for 5 min at 95° C. Fluorescent dye-labelled protein mixtureswere analyzed by SDS-PAGE as described above.

Example 14 Construction of Mammalian Cell Expression Vector

For expression of fusion proteins ^(N)AGT-NLS3, ^(N)AGT-βGal, ACT1-NLS3and ACT1 βGal in mammalian cells, ^(N)AGT and ACT1 genes were PCRamplified using primers 8 (N, AGT, SEQ ID NO:10) and 9 (C, AGT, SEQ IDNO:11) and inserted into NheI/BglII restriction sites of the mammalianexpression vector pECFP-Nuc (Clontech) or a mammalian expression plasmidcontaining the β-galactosidase gene.

The invention claimed is:
 1. A recombinant alkylcytosine transferase(ACT) comprising a protein having at least one cysteine capable ofreacting with an O₂-benzylcytosine, thereby transferring the benzylsubstituent to the mercapto function of the cysteine at least as fast ason reaction with an O₆-benzylguanine under identical conditions; andwherein (i) the ACT protein has the amino acid sequence of SEQ ID NO:1or SEQ ID. No 12; or (ii) the ACT protein differs from SEQ ID. No 1 byone or more amino acid substitutions selected from the groups consistingof: R114A, S131V, E148Q, G157W, M159R; R114S, S131T, D135T, E148D,G157P, M159E; R114N, S131N, G157A, M159S; R114A, S131T, D135S, G157K,M159E; R114E, S131R, D135A, G157E, M159E; R114S, S131V, E148Q, G157L,M159R; R114E, S131N, D135N, G157T, M159F; and wherein the variantdiffers from SEQ ID NO: 1 or SEQ ID NO: 12 in one, two or three aminoacids in positions other than positions 114, 131, 135, 148, 157, and159; or wherein (iii) the variant differs from SEQ ID. No 1 or 12 by anamino acid in position 60 is M or I; an amino acid in position 114 is A,E, N, R or S; an amino acid in position 121 is A or V; an amino acid inposition 131 is N, S, T or V; an amino acid in position 135 is D, N orT; an amino acid in position 148 is D, E, Q or V; an amino acid inposition 153 is L or S; an amino acid in position 157 is A, G, L, T, Por W; and an amino acid in position 159 is E, F, M, R, S or L; or (iv)the variant differs from SEQ ID: 12 by one, two or three amino acids ata position other than positions 60, 114, 121, 131, 135, 148, 153, 157,and
 159. 2. A method for detecting and/or manipulating a protein ofinterest, comprising: (a) creating a fusion protein comprising a proteinof interest and a variant of an alkylcytosine transferase according toclaim 1; (b) contacting the alkylcytosine transferase fusion proteinwith a compound of formula (I)

wherein R1 is an aromatic or a heteroaromatic group, or an optionallysubstituted unsaturated alkyl, cycloalkyl or heterocyclyl group with thedouble bond connected to OCH2—; R2 is a linker; and L is a label or aplurality of same or different labels; and (c) detecting the fusionprotein and optionally further manipulated using the label L in a systemdesigned for recognising and/or handling the label.